T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends

Nucleic Acids Res. 2003 Jun 15;31(12):e68. doi: 10.1093/nar/gng068.

Abstract

Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy--T-linker-specific ligation PCR (T-linker PCR) for gene or chromosome walking. The strategy amplifies the template molecules in three steps. First, genomic DNA is digested with 3' overhang enzymes. Secondly, primed by a specific primer, a strand of the target molecule is replicated by Taq DNA polymerase and a single A tail is generated on the 3' unknown end of the target molecule, and then a 3' overhang-T linker (named T-linker) is specifically ligated onto the target. Thirdly, the target is amplified by two rounds of nested PCR with specific primers and T-linker primers. T-linker PCR significantly improves the existing PCR methods for walking because it uses specific T/A ligation instead of arbitrary ligation or random annealing. To show the feasibility and efficiency of T-linker PCR, we have exploited this method to identify vector DNA or T-DNA insertions in transgenic plants.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Capsid Proteins / genetics
  • Chromosome Walking / methods*
  • DNA / isolation & purification*
  • DNA Restriction Enzymes
  • DNA, Bacterial / analysis
  • Data Interpretation, Statistical
  • Genes, Viral
  • Genome, Plant
  • Genomics / methods
  • Mutagenesis, Insertional
  • Mutation
  • Oryza / genetics
  • Plants, Genetically Modified
  • Polymerase Chain Reaction / methods*
  • Templates, Genetic

Substances

  • Capsid Proteins
  • DNA, Bacterial
  • T-DNA
  • S8 protein, rice gall dwarf virus
  • DNA
  • DNA Restriction Enzymes